Although 26 years has passed since HIV-1 was identified as the causative agent for AIDS, the percentage of people living with HIV has steadily increased as new infections occur each year and as HIV treatments extend life. An estimated 33 million people were living with HIV in 2007. Most of the current drugs in use for the treatment of AIDS work by combination targeting of the enzymatic activities of the HIV reverse transcriptase and/or protease (HAART, highly active anti-retroviral therapy) and/or gp41. These treatments remain expensive and are often not well- tolerated by patients. Because of the emergence of drug-resistant virus that commonly occurs as the result of classical HIV treatment, there remains a great need to continue the search for alternative therapies that target other essential viral activities. Thus, the development of new drugs with novel mode of action is of utmost urgency. We propose to develop inhibitors of protein-RNA interactions that are absolutely essential for the HIV life cycle-excellent targets widely recognized by the community. TAR and RRE RNAs are well-conserved noncoding sequences in the viral genome with defined targetable structures. Targeting these RNA structures is attractive because the affect virus-specific interactions that could potentially lead to specific inhibition of viral replication, with possibly minimal side effects on other cellular functions. Additionally, this strategy can result in a lower incidence of drug resistance since the regulation of HIV-1 transcription requires the interplay of both viral and cellular components. We have recently reported that branched peptides are good selective ligands for HIV-1 TAR. We propose to carry-out the screening of combinatorial libraries of short branched peptides. This is accomplished with specific aim 1, wherein a larger library of diversified branched peptides and borono-branched peptides is synthesized. Because the major challenge in targeting RNA is imparting selectivity to a particular sequence/structure, a highly stringent screening assay is necessary. This is also addressed in Aim 1. We recognize that during a high throughput screening assay, false positives are possible and that an RNA binder does not necessarily indicate perturbation of the desired protein/RNA interaction. In aim 2, we use a series of cell based assays to test the inhibitory activities of hit branched peptides against HIV-1 Tat/TAR and Rev/RRE interactions. Additionally, we investigate the selectivity as well as possible cytotoxicity of branched peptide inhibitors. PUBLIC HEALTH RELEVANCE: The proposed research aims to discover and develop medium-sized molecules that selectively inhibit the interaction of protein-RNA interactions that are absolutely essential for HIV-1 viral replication. If successful, these molecules will be the next generation drugs with novel mode of action as anti-HIV therapy.